Expansions of CAG trinucleotide repeats (CAG repeats) in coding regions of human genes cause numerous disorders by generating proteins with elongated polyglutamine (polyQ) stretches. This group of disorders includes by way of example Dystophia myotonica, Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 17, Huntington disease-like 2, Spinal and bulbar muscular atrophy, Huntington disease, Dentatorubral-pallidoluysian atrophy, Oculopharyngeal dystrophy, Congenital central hypoventilation syndrome, Infantile spasms, Synpolydactyl), Cleidocranial dysplasia, Holoprosencephaly, Hand-foot-genital syndrome, Type II blephorophimosis, ptosis, and epicanthus inversus syndrome. (Wanker E. E. (2000) Biol. Chem., 381:937-942; Gusella J. F. and MacDonald, M. E. (2000) Nature Rev. Neurosci., 1:109-115; and Usdin K. and Grabczyk, E. (2000) Cell. Mol. Life. Sci., 57:914-931).
For purposes of illustration only Huntington's disease (HD) will be discussed herein. HD is a chronic neurodegenerative disorder which is inherited as an autosomal dominant trait and is characterized by chorea, dementia and personality disorder. Martin, J. B. and Gusella, J. F. (1986) N. Engl. J. Med. 315:1267-1276. The gene responsible for HD contains an expanded and unstable CAG trinucleotide repeat. Huntington's Disease Collaborative Research Group (1993) Cell 72:971-983.
The HD gene (IT15 gene), which encodes huntingtin, a 350 kDa protein of unknown function, is located on the human chromosome 4 and consists of 67 exons. The disease-causing mutation is a CAG repeat expansion located within exon 1 of the HD gene (HD exon1). The CAG repeat is translated into a polyQ stretch. The disease manifests itself when the polyQ stretch exceeds the critical length of 37 glutamines (pathological threshold), whereas 8-35 glutamine residues in huntingtin are tolerated by neuronal cells. Experimental evidence has been presented that huntingtin fragments with polyQ tracts in the pathological range (more than 37 glutamines), but not in the normal range (20-32 glutamines), form high molecular weight protein aggregates with a fibrillar morphology in vitro and in cell culture model systems (Scherzinger et al. (1999) Proc. Natl. Acad. Sci. USA, 96:4604-4609; and Waelter et al., (2001) Mol. Biol. Cell, 12:1393-1407). In addition, inclusions with aggregated N-terminally truncated huntingtin protein were detected in HD transgenic mice carrying a CAG repeat expansion of 115-156 units and in HD patient brains (Davies et al., (1997) Cell, 90:537-548; and DiFiglia et al., (1997) Science, 277:1990-1993), suggesting that the process of aggregate formation may be important for the progression of HD. The mechanisms, however, by which the elongated polyQ sequences in huntingtin cause dysfunction and neurodegeneration are not yet understood (Scherzinger et al., (1999); Tobin A. J. and Signer, E. R. (2000) Trends Cell Biol., 10:531-536; and Perutz M. F. (1999) Glutamine repeats and neurodegenerative diseases: molecular aspects. Trends Biochem. Sci., 24:58-63).
Unaffected individuals have repeat numbers of up to 30, while individuals at a high risk of developing HD carry more than 37 CAG repeats. Individuals with 30-37 repeats have a high risk of passing on repeats in the affected size range to their offspring (Andrew et al., (1997) Hum. Mol. Genet., 6:2005-2010; Duyao et al., (1993) Nature Genet., 4:387-392; and Snell et al., (1993) Nature Genet., 4:393-397).
It is known that patients are able to survive and live healthy lives with only one functioning copy of the IT15 gene. Thus, selective inactivation of the allele with a disease-causing mutation should diminish or even eliminate the disease while improving the possibilities of survival in heterozygous patients.
The combination of emotional, cognitive and motor symptoms in HD contributes to an unusually high cost of care. People with Huntington's Disease require care from health professionals of many stripes, including general practitioners, neurologists, social workers, home health aides, psychologists, physical therapists, and speech/language pathologists.
Currently, there are a few diagnostic approaches for nucleic acid sequence identification. U.S. Patent Application Publication No. 20040048301 describes allele-specific primer extension in the presence of labeled nucleotides for sequence identification, but does not include allele-specific primer extension for enrichment of one allele over the other for further analysis of the allele of interest as part of the kit. WO Patent Application No. 2003100101 describes isolation of one sequence in a mixture by hybridization markers and single-strand specific nucleases for use in single-molecule analysis. U.S. Patent Application Publication No. 20030039964 describes a method for isolation of one sequence in a mixture by hybridization to a fixed probe, but does not disclose the use of reverse transcription. U.S. Pat. No. 6,013,431 describes a method for analysis of bases adjacent to a hybridized, immobilized oligo, but does not disclose enrichment of one allele over the other. WO Patent Application No. 9820166 describes a method for specific selection of one allele over the other, followed by mass spectroscopic analysis of the selected molecule, but does not disclose the use of reverse transcription. None of these references disclose methods and diagnostic kits for linking polymorphic sequences to expanded repeat mutations for improved allele-specific diagnosis.
U.S. Patent Publication No. 20040241854 (Davidson) discloses allele-specific inhibition of specific single nucleotide polymorphism variants, and presents data showing that “expanded CAG repeats and adjacent sequences, while accessible to RNAi, may not be preferential targets for silencing” thus describing the problem that our invention addresses (determining what SNP variant at a remote molecular position is linked to the expanded CAG repeat in a particular patient), but does not teach the use of reverse transcription using an allele-specific primer to solve this problem, nor otherwise disclose a method for how to solve this problem. U.S. Patent Publication No. 20060270623 (McSwiggen) discloses multiple siRNA sequences, including those comprising SNP variants, but does not provide any working examples regarding allele-specific RNA interference using these disclosed siRNA sequences, nor disclose how to determine which allele-specific siRNA to administer to a particular Huntington's disease human patient in order to effectively treat that patient's disease by suppression of only the expanded Huntington allele in that patient.
Accordingly, there is need in the art for novel compounds, methods, and kits for allele-specific diagnostics and therapies.